Abstract 5697: Targeting CBL and CBLB ubiquitin ligases to exhaust cancer stem cells in metastatic breast cancer

Abstract Introduction: Recurrence, therapy resistance and metastasis remain major challenges in cancer therapy, accounting for nearly all deaths from cancer. The inability of current therapies to eliminate cancer stem cells (CSCs) is widely believed to underlie these challenges. Targeting molecular pathways that help maintain the CSCs could open new avenues to devise durable cancer therapies. The CBL-family E3 ubiquitin ligases (CBL, CBL-B and CBL-C) attenuate tyrosine kinase signaling through ubiquitin-dependent degradation of tyrosine kinase-coupled receptors and signaling components. Gene deletion studies have shown that CBL-B, the predominant family member expressed in mature immune cells, mediates tumor-induced “immune editing” to generate a pro-tumor immune microenvironment. This mechanism has provided one line of evidence to develop chemical CBL-B inhibitors as cancer immune modulators, and candidate inhibitors exhibit anti-tumor activity in mouse implant models, supporting the general idea that CBL-family E3s are potential therapeutic targets. Using mouse genetic models, we have established that CBL and CBL-B are redundantly required for the maintenance of adult stem cell compartments, and that this role is dependent on the core function of CBL/CBL-B as negative regulators of the AKT-mTOR axis downstream of tyrosine kinase signaling. As normal organ stem cell programs are frequently co-opted by CSCs; we used a 3-pronged approach to ask if CBL/CBL-B are required to maintain CSC function. Methods/Results: 1. Based on higher CBL/CBL-B expression in TNBC cell lines, and TCGA data that CBL/CBL-B mRNA expression positively correlates with shorter patient survival, we engineered Dox-inducible shRNAs in TNBC cell lines and showed that CBL/CBL-B KD impaired cell migration and invasion, reduced the population with CSC markers and impaired orthotopic xenograft tumorigenesis and metastatic seeding. 2. We generated a unique mouse model in which the C3(1)-TAg transgene drives mammary tumors known to resemble human triple-negative breast cancer (TNBC) and floxed-Cbl and Cblb genes can be inducibly deleted using tamoxifen (via CreERT2). Tumors of these mice established as tumor-derived organoids (TDOs) exhibited significant impairment of growth and CSC signature gene expression upon Cbl/Cblb KO. 3. Since the developed “CBL-B” inhibitors target a mechanism shared between CBL/CBL-B, we used two active compounds (NTX-801, NTX-944) and one inactive compound (NTX-616) developed by Nimbus Therapeutics. The active inhibitors mimicked the genetic Cbl/Cblb KO to inhibit the mouse mammary TDOs growth, while the inactive analog was without effect. Conclusions: Our results establish a tumor-intrinsic requirement of CBL/CBL-B to maintain breast cancer CSCs and add a critical second rationale for the use of chemical inhibitors of CBL/CBL-B as targeted therapeutics to counter BC recurrence and metastasis. Citation Format: Bhopal C. Mohapatra, Aaqib M. Bhat, Mohsin Raza, Haitao Luan, Santosh Shrestha, Sowmya Kolluru, Matthew D. Storck, Lusheng Li, Fu-Shan Kuo, Silvana Leit, Fang Qiu, Subodh M. Lele, David Ciccone, Christine Loh, Shibiao Wan, Vimla Band, Hamid Band. Targeting CBL and CBLB ubiquitin ligases to exhaust cancer stem cells in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5697.